Method of inhibiting abcg2 and related treatments

ABSTRACT

Disclosed are methods of enhancing the chemotherapeutic treatment of tumor cells, reducing resistance of a cancer cell to a chemotherapeutic agent, a method of inhibiting ABCG2 or MRP1 in a mammal afflicted with cancer, and a method of increasing the bioavailability of an ABCG2 substrate drug in a mammal. The methods comprise administering peliomycin and other compounds described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 12/742,036, filed Jun. 23, 2010, which is a U.S.National Phase of International Patent Application No.PCT/US2008/082414, filed Nov. 5, 2008, which claims the benefit of U.S.Provisional Patent Application No. 60/986,155, filed Nov. 7, 2007, allof which are incorporated by reference.

BACKGROUND OF THE INVENTION

Multidrug resistance has long been recognized as a major obstacle tosuccessful cancer chemotherapy. The multidrug resistance transporterABCG2 (or Breast Cancer Resistance Protein 1, BCRP1), a member of theABC (ATP-binding cassette) family of membrane transport proteins, isbelieved to form a part of the maternal-fetal barrier, the blood-brainbarrier, and is known to limit oral absorption of some drugs (Robey etal., Cancer Metastasis Rev., 26: 39-57 (2007)). The normal physiologicfunctions of ABCG2 may be related to transport of a variety of naturalsubstances to prevent intracellular accumulation of toxic compounds.ABCG2 is also an important mediator of resistance to a variety ofanti-cancer drugs, including mitoxantrone, topotecan, irinotecan,flavopiridol, and methotrexate (Sarkadi et al., Physiol. Rev., 86:1179-1236 (2006); Krishnamurthy et al., Annu. Rev. Pharmacol. Toxicol.,46: 381-410 (2006); Szakacs et al., Nat. Rev. Drug Discov., 5: 219-34(2006); and Xu et al., Curr. Med. Chem., 14: 689-701 (2007)). Thus,inhibitors of ABCG2 activity could have important oncologic andpharmacologic applications.

Unfortunately, few, if any, clinically useful inhibitors of ABCG2activity have been reported. Thus, there exists a desire for compoundsthat can inhibit ABCG2 and in turn, increase the efficacy of adjuvantchemotherapy.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of enhancing the chemotherapeutictreatment of tumor cells in a mammal with a chemotherapeutic agent,which method comprises administering to the mammal an effective amountof the chemotherapeutic agent in conjunction with an effective amount ofa compound described herein that inhibits ABCG2 protein.

In an embodiment, the invention provides a method of reducing resistanceof a cancer cell to a chemotherapeutic agent by inhibiting ABCG2 in amammal, which method comprises administering to the mammal an effectiveamount of a compound described herein.

The invention further provides a method of inhibiting ABCG2 and/or MRP1in a mammal afflicted with cancer, which method comprises administeringto the mammal an effective amount of a compound described herein.

The invention also provides a method of increasing the bioavailabilityof an ABCG2 substrate drug in a mammal, which method comprisesadministering to the mammal an effective amount of the ABCG2 substratedrug in conjunction with an effective amount of a compound describedherein that inhibits ABCG2 protein.

The invention further provides use of an ABCG2 inhibitor or an MRP1inhibitor in the preparation of a medicament for enhancing thechemotherapeutic treatment of a mammal afflicted with cancer. Theinvention also provides use of an ABCG2 inhibitor or an MRP1 inhibitorin combination with a chemotherapeutic agent for treating cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts the activity of compounds in a pheophorbide a (PhA)screening assay in an embodiment of the invention. The compounds areassayed for their ability to cause ABCG2-overexpressing cells(NCI-H460/MX20 cells) to accumulate PhA. Each compound is tested at 10μM (final concentration—bars) and, after resupply, in a dose-responseformat. Activity for each compound is normalized to the response of 10μM FTC control wells on the same plate. Error bars represent sem(n=7-9). IC₅₀ values are averages of duplicate determinations for eachdose.

FIG. 2 depicts the sensitization of ABCG2-overexpressing cells tomitoxantrone in an embodiment of the invention. Cell survival isnormalized to buffer control (no mitoxantrone, no compound=100%). Errorbars represent range of duplicate determinations. The white barsindicate mitoxantrone administration alone, whereas the gray barsindicate administration of 30 μM mitoxantrone and a compound of theinvention.

FIGS. 3A-3C depict functional assays of the effects of compounds onABCG2 and MRP1 in an embodiment of the invention. Column 1 in FIG.3A-3C: ABCG2 transfected cells are incubated in BODIPY-prazosin in theabsence (shaded histogram) or presence of 0.1 (heavy solid line), 1(solid line), or 10 μM (dashed line) of NSC11668, NSC19139, NSC120688,NSC168201 or NSC375985. FTC (10 μM, bottom histogram) is shown as apositive control for ABCG2 inhibition. Column 2 in FIG. 3A-3C: ABCG2transfected cells are incubated with unlabeled 5D3 antibody (1:3500) inthe absence (solid line) or presence (dashed line) of 20 μM of acompound after which cells are incubated in APC-labeled secondaryantibody. FTC (bottom histogram) at a concentration of 20 μM is shown asa positive control. Column 3 in FIG. 3A-3C: MDR1-transfected cells areincubated in 0.5 μg/ml rhodamine 123 in the absence (solid line) orpresence (dashed line) of 10 μM of the desired inhibitor. Valspodar at 3μg/ml is included as a positive control for Pgp inhibition (bottomhistogram). Column 4 in FIG. 3A-3C: MRP1-transfected cells are incubatedin 200 nM calcein AM in the absence (solid line) or presence (dashedline) of 10 μm of the compounds. MK-571 (25 μM) is shown (bottomhistogram) as a positive control for inhibition of MRP1 transport.

FIG. 4 depicts the amount of [¹²⁵I]-IAAP incorporated (average of threeindependent experiments) into ABCG2 (Y-axis) in the absence (control) orpresence of 20 μM of the indicated compound (X-axis).

FIGS. 5A-5F depict the effect of ABCG2 inhibitor on control and ABCG2transfected cells when treated with varying concentrations ofchemotherapeutic drug SN-38, which is 7-ethyl-10-hydroxycamptothecin.The Y-axis represents the optical density of cell suspension (arbitraryunits) and the X-axis represents the concentration of thechemotherapeutic drug, SN-38. Squares and diamonds represent positivecontrol HEK 293 cells, with squares representing experiments without theABCG2 inhibitor and the diamonds representing experiments with the ABCG2inhibitor. Triangles and circles represent HEK 293 cells transfectedwith ABCG2, with triangles representing experiments conducted with theABCG2 inhibitor, and the circles representing experiments without theABCG2 inhibitor.

FIG. 5A depicts the effect where the ABCG2 inhibitor is NSC 11668. FIG.5B depicts the effect where the ABCG2 inhibitor is NSC 19139. FIG. 5Cdepicts the effect where the ABCG2 inhibitor is NSC 120688. FIG. 5Ddepicts the effect where the ABCG2 inhibitor is NSC 168201. FIG. 5Edepicts the effect where the ABCG2 inhibitor is 375985. FIG. 5F depictsthe effect where the ABCG2 inhibitor is FTC (fumitremorgin C).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in accordance with an embodiment, a method ofenhancing the chemotherapeutic treatment of tumor cells in a mammal witha chemotherapeutic agent, which method comprises administering to themammal an effective amount of the chemotherapeutic agent in conjunctionwith an effective amount of a compound to inhibit ABCG2 protein, saidcompound being selected from the group consisting of peliomycin (NSC76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A. A can be anysuitable mono- or divalent anion (e.g., a Group VII anion, such as Br⁻,Cl⁻, or I⁻).

By “enhancing the chemotherapeutic treatment” is meant that thechemotherapeutic agent has a greater effect (e.g., at least a 5%increase, at least a 10% increase, at least a 20% increase, at least a30% increase, at least a 40% increase, at least a 50% increase, at leasta 60% increase, at least a 70% increase, at least an 80% increase, etc.)in the presence of at least one compound described herein than in theabsence of that compound. Since ABCG2 is a mediator of resistance, if acompound described herein inhibits ABCG2, the cancerous cell is lessresistant to the chemotherapeutic agent, thereby making it moresusceptible to the cytotoxicity of the agent.

The present invention also provides a method of reducing resistance of acancer cell to a chemotherapeutic agent by inhibiting ABCG2 in a mammal,which method comprises administering to the mammal an effective amountof a compound selected from the group consisting of peliomycin (NSC76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A, whereuponresistance of the chemotherapeutic agent is reduced in the mammal. By“reducing resistance of a chemotherapeutic agent” is meant that cancercells that are treated by the chemotherapeutic agent have resistancereversed, development of resistance is reduced, or a combinationthereof. For example, resistance is reduced by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, or at least 80%.

The chemotherapeutic agent described herein can be any cytotoxic drugthat is useful to kill cancer cells. In embodiments, the agent is anydrug in which there is resistance to a cancer cell upon administration.For example, the agent can be an antimetabolite (e.g., methotrexate), amitotic inhibitor (e.g., docetaxel, paclitaxel, vinblastine), analkylating agent (e.g., cisplatin), a cytotoxic antibiotic (e.g.,daunorubicin, doxorubicin, mitoxantrone), a topoisomerase inhibitor(e.g., topotecan, irinotecan, camptothecin, SN-38), a tyrosine kinaseinhibitor (e.g., gefitinib), or any combination thereof. Specificexamples of the chemotherapeutic agent include mitoxantrone, topotecan,irinotecan, flavopiridol, gefitinib, methotrexate, rhodamine,daunomycin, imatinib, doxorubicin, colchincine, vinblastine, paclitaxel,cisplatin, adriamycin, danofloxacin mesylate, and/or docetaxel. In someembodiments, the chemotherapeutic agent is mitoxantrone, topotecan,irinotecan, flavopiridol, gefitinib, methotrexate, or any combinationthereof.

Any method known in the art can be used to measure the enhancement ofthe chemotherapeutic agent and/or the reduction of resistance. TheExamples section describes exemplary methods. Alternatively, cells canbe contacted with a toxic chemotherapy drug, such as mitoxantrone ortopotecan, in an amount that permits cell survival due to the resistanceconferred by ABCG2. Cell viability can be measured by a colorimetricassay (Skehan et al., J. Natl. Cancer Inst. 82: 1107 1112 (1990)), bycounting cells with a cell counter, or by incorporation of tritiatedthymidine.

The cells are then contacted with a compound of the invention thatinhibits ABCG2. The enhancement of the chemotherapeutic agent and/orreduction of resistance can then be detected by measuring the growthinhibition of cells, using a variety of means, such as IC₅₀measurements, vital staining, metabolite measurements, or confocalmicroscopy. Confocal microscopy can be used to determine whether aparticular drug has been retained or accumulated in the cell.

The invention further provides a method of inhibiting ABCG2 in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound selected from the groupconsisting of peliomycin (NSC 76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A, whereuponABCG2 is inhibited in the mammal.

Since ABCG2 has also been reported to be expressed at high levels in thedigestive tract and at the blood-brain barrier (Takano et al.,Pharmacol. Ther., 109: 137-61 (2006)), it is envisioned that ABCG2inhibitors can enhance bioavailability (e.g., oral bioavailability) andbrain penetration of ABCG2 substrate drugs, such as, e.g., topotecan.Thus, in an embodiment, the present invention provides a method ofincreasing the bioavailability of an ABCG2 substrate drug in a mammal,which method comprises administering to the mammal an effective amountof the ABCG2 substrate drug in conjunction with an effective amount of acompound to inhibit ABCG2 protein, said compound being selected from thegroup consisting of peliomycin (NSC 76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A. Since CNSpenetration can be enhanced by administration of a compound of theinvention, such method is useful in the treatment of cancer, such asbrain tumors, CNS metastases, and/or gastrointestinal stromal tumors.

The ABCG2 substrate drug can be, for example, an antimetabolite (e.g.,methotrexate), a mitotic inhibitor (e.g., docetaxel, paclitaxel,vinblastine), an alkylating agent (e.g., cisplatin), a cytotoxicantibiotic (e.g., daunorubicin, doxorubicin, mitoxantrone), atopoisomerase inhibitor (e.g., topotecan, irinotecan, camptothecin,SN-38), a tyrosine kinase inhibitor (e.g., gefitinib), or anycombination thereof. Specific examples of the ABCG2 substrate druginclude mitoxantrone, topotecan, irinotecan, flavopiridol, gefitinib,methotrexate, rhodamine, daunomycin, imatinib, doxorubicin, colchincine,vinblastine, paclitaxel, cisplatin, adriamycin, danofloxacin mesylate,docetaxel, or any combination thereof. In some embodiments, the ABCG2substrate drug is mitoxantrone, topotecan, irinotecan, flavopiridol,gefitinib, and/or methotrexate.

Pgp, MRP1, and ABCG2 are the major contributors to multidrug resistancein most cancer cells in culture (Szakacs et al., Nat. Rev. Drug Discov.,5: 219-34 (2006)). ABCG2 has overlapping substrate specificity with MRP1and Pgp. Accordingly, in an embodiment, the present invention provides amethod of inhibiting MRP1 in a mammal afflicted with cancer, whichmethod comprises administering to the mammal an effective amount of acompound selected from the group consisting of

and any combination thereof, whereupon MRP1 is inhibited in the mammal.

In preferred embodiments of the methods described herein, the compoundis selected from the group consisting of

and any combination thereof.

All of the methods described herein have applicability to the treatmentof any type of cancer that over-expresses ABCG2 (and/or MRP1) and iscapable of being treated with a chemotherapeutic agent. Such cancersinclude, for example, leukemias (e.g., acute myeloid leukemia (AML),chronic myeloid leukemia (CML)), solid tumors (e.g., of the lung,endometrium, or digestive tract), melanomas, non-small cell lung cancertumors, colon tumors, prostate tumors, brain tumors, lymphomas, breasttumors, ovarian tumors, lung tumors, and stomach tumors.

For purposes of the present inventive methods, the mammal includes,without limitation, the order Rodentia, such as mice, and the orderLogomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). It is most preferred that themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). An especially preferred mammalis the human.

The methods of the present invention encompass administration of apharmaceutical composition comprising (i) a compound selected from thegroup consisting of peliomycin (NSC 76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A and (ii) apharmaceutically acceptable carrier.

Generally, the compounds of the invention will be administered in apharmaceutical composition to an individual afflicted with a cancer.Those undergoing or about to undergo chemotherapy can be treated with atleast one compound described herein separately or in conjunction withother treatments, as appropriate. In therapeutic applications,compositions are administered to a patient in an amount sufficient toelicit an effective depression of ABCG2 activity thereby potentiatingthe cytotoxicity of the chemotherapeutic treatment. A dose adequate toaccomplish this is defined as an “effective amount,” which is also an“ABCG2 inhibiting effective amount.” Amounts effective for a therapeuticor prophylactic use will depend on, e.g., the stage and severity of thedisease being treated, the age, weight, and general state of health ofthe patient, and the judgment of the prescribing physician. The size ofthe dose will also be determined by the compound selected, method ofadministration, timing and frequency of administration as well as theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound and the desiredphysiological effect. It will be appreciated by one of skill in the artthat various disease states may require prolonged treatment involvingmultiple administrations, perhaps using a series of different ABCG2inhibitors and/or chemotherapeutic agents in each or various rounds ofadministration.

Suitable chemotherapeutic agents administered in coordination with atleast one compound of the present invention include mitoxantrone,topotecan, irinotecan, flavopiridol, gefitinib, methotrexate, rhodamine,daunomycin, imatinib, doxorubicin, colchincine, vinblastine, paclitaxel,cisplatin, adriamycin, danofloxacin mesylate, camptothecin, SN-38,and/or docetaxel. The chemotherapeutic agent is administered in a dosesufficient to treat the cancer (e.g., cancer-treatment effective amountof a chemotherapeutic agent). Such doses are known in the art (see, forexample, the Physicians' Desk Reference (2004)). Such agents can beadministered using techniques such as those described in, for example,Wasserman et al., Cancer, 36, pp. 1258-1268 (1975) and Physicians' DeskReference, 58th ed., Thomson PDR (2004).

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound of the present invention.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. The present inventivemethods can involve the administration of about 0.1 μg to about 50 mg ofat least one compound of the invention per kg body weight of theindividual. For a 70 kg patient, dosages of from about 10 μg to about200 mg of the compound of the invention would be more commonly used,depending on a patient's physiological response, e.g., as determined bymeasuring cancer-specific antigens or other measurable parametersrelated to the tumor load of a patient.

Any of the compound of the invention is administered in a dosesufficient to enhance the effect of the chemotherapeutic agent and/orreduce drug resistance in a cancer. A suitable dosage is that which willresult in a concentration of the compound of the invention in thecancerous cells to be treated sufficient to inhibit ABCG2 activity,e.g., from about 10 nM to 200 nM intracellularly, which can require anextracellular concentration of from about 10 μM to 50 μM. The dose canbe adjusted as necessary to enhance the effect of the chemotherapeuticagent and/or reduce drug resistance.

The pharmaceutical compositions for therapeutic treatment are intendedfor any suitable mode of administration, including parenteral, topical,oral, or local administration and generally comprise a pharmaceuticallyacceptable carrier and an amount of the active ingredient sufficient toreduce, and preferably prevent, the activity of ABCG2. The carrier canbe any of those conventionally used and is limited only bychemico-physical considerations, such as solubility and lack ofreactivity with the compound of the invention, and by the route ofadministration.

The pharmaceutically acceptable carrier (or excipient) is preferably onethat is chemically inert to the compound of the invention and one thathas no detrimental side effects or toxicity under the conditions of use.Such pharmaceutically acceptable carriers preferably include saline(e.g., 0.9% saline), Cremophor EL (which is a derivative of castor oiland ethylene oxide available from Sigma Chemical Co., St. Louis, Mo.)(e.g., 5% Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90%saline, or 50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40%propylene glycol/10% ethanol/50% water), polyethylene glycol (e.g., 40%PEG400/60% saline), and alcohol (e.g., 40% ethanol/60% water). Apreferred pharmaceutical carrier is polyethylene glycol, such as PEG400, and particularly a composition comprising 40% PEG 400 and 60% wateror saline. The choice of carrier will be determined in part by theparticular compound chosen, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention.

The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, rectal, andvaginal administration are merely exemplary and are in no way limiting.The pharmaceutical compositions can be administered parenterally, e.g.,intravenously, intraarterially, subcutaneously, intradermally,intrathecally, or intramuscularly. Thus, the invention providescompositions for parenteral administration that comprise a solution ofthe compound of the invention dissolved or suspended in an acceptablecarrier suitable for parenteral administration, including aqueous andnon-aqueous, isotonic sterile injection solutions.

Preferably a compound of the invention and a chemotherapeutic agent arecoadministered to the mammal. By “coadministering” is meantadministering the chemotherapeutic agent and a compound of the inventionsufficiently close in time such that a compound of the invention canenhance the effect of the chemotherapeutic agent. In this regard, acompound of the invention can be administered first and thechemotherapeutic agent can be administered second, or vice versa.Alternatively, a compound of the invention and the chemotherapeuticagent can be administered simultaneously. In addition, a combination ofcompounds of the invention can be administered, and one or more of thecompounds of the invention can be administered in combination withanother agent useful in the treatment of cancer.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986). Such compositions include solutions containing anti-oxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. The compounds of theinvention can be administered in a physiologically acceptable diluent ina pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol (for example intopical applications), or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, suchas 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, and synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral oil. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylene polypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations typically will contain from about 0.5% orless to about 25% or more by weight of a compound of the invention insolution. Preservatives and buffers can be used. In order to minimize oreliminate irritation at the site of injection, such compositions cancontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of the present invention for application toskin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the inventiondissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apre-determined amount of the compound of the invention, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations can include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and cornstarch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising a compound of theinvention in an inert base, such as gelatin and glycerin, or sucrose andacacia, emulsions, gels, and the like containing, in addition to thecompound of the invention, such excipients as are known in the art.

A compound of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. A compound of the invention is preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of the compounds of the invention can be about 0.01%to about 20% by weight, preferably about 1% to about 10% by weight. Thesurfactant must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such surfactants are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides canbe employed. The surfactant can constitute from about 0.1% to about 20%by weight of the composition, preferably from about 0.25% to about 5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin, for intranasal delivery.These aerosol formulations can be placed into acceptable pressurizedpropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also can be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations can be used to spray mucosa.

Additionally, a compound of the invention can be made into suppositoriesby mixing with a variety of bases, such as emulsifying bases orwater-soluble bases. Formulations suitable for vaginal administrationcan be presented as pessaries, tampons, creams, gels, pastes, foams, orspray formulas containing, in addition to the active ingredient, suchcarriers as are known in the art to be appropriate.

The concentration of a compound of the present invention in thepharmaceutical formulations can vary, e.g., from less than about 1%,usually at or at least about 10%, to as much as 20% to 50% or more byweight, and can be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 100mg of at least one compound of the invention. Actual methods forpreparing parenterally administrable compounds of the invention will beknown or apparent to those skilled in the art and are described in moredetail in, for example, Remington's Pharmaceutical Science (17th ed.,Mack Publishing Company, Easton, Pa., 1985).

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, a compoundof the invention can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes. Liposomes can serve totarget a compound of the invention to a particular tissue, such aslymphoid tissue or cancerous hepatic cells. Liposomes can also be usedto increase the half-life of a compound of the invention. Many methodsare available for preparing liposomes, as described in, for example,Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

Pheophorbide a (PhA) is obtained from Frontier Scientific (Logan, Utah).Fumetrimorgin C (FTC), libraries of pure natural products, and syntheticmolecules (structural diversity and mechanistic diversity sets) andindividual compounds are from the Drug Synthesis and Chemistry Branch,Developmental Therapeutics Program (DTP), Division of Cancer Treatmentand Diagnostics, National Cancer Institute (Bethesda, Md.). Cell culturemedia are from Invitrogen (Carlsbad, Calif.), fetal bovine serum (FBS)from Hyclone (Kansas City, Mo.), and phosphate-buffered saline (PBS)from Quality Biological (Gaithersburg, Md.).

Intracellular fluorescence of BODIPY-prazosin, rhodamine 123, or calceinfluorescence is detected with a FACSort flow cytometer equipped with a488 nm argon laser and 530 nm bandpass filter. APC fluorescence ismeasured with a 635 nm read diode laser and 561 nm longpass filter. Atleast 10000 events are collected. Dead cells are eliminated based onpropidium iodide exclusion.

Apparent IC₅₀ values are calculated from dose-response data usingSigmaPlot (SPSS, Inc., Chicago) 4-parameter logistic nonlinearregression analysis. Unless otherwise noted, all data are presented asaverage±sem.

EXAMPLE 1

This example demonstrates a screening assay for ABCG2 inhibitors inaccordance with an embodiment of the invention.

To prepare the cell culture, NCI-H460 human lung non-small-cellcarcinoma cells (National Cancer Institute, Frederick, Md.) are selectedfor over-expression of ABCG2 by maintenance in RPMI1640/10% FBSsupplemented with 20 nM mitoxantrone (Robey et al., Biochim. Biophys.Acta, 1512: 171-82 (2001)). After removal of mitoxantrone, cells arefurther grown in the same medium without mitoxantrone for 5-30 days.These cells are designated NCI-H460/MX20. Parental cells (low ABCG2expression) (Robey et al., vide supra) are maintained in the same mediumwithout mitoxantrone. ABCG2-transfected or MDR1-transfected (i.e.Pgp-expressing) HEK293 cells are maintained in 2 mg/ml G418 aspreviously described (Robey et al., Br. J. Cancer, 89: 1971-8 (2003)).MRP1-transfected HEK293 cells are maintained in 5 μM etoposide. MCF-7FLV1000 cells are maintained in Richter's medium with 10% FCS andPen/strep with 1000 nM flavopiridol (Robey et al., Clin. Cancer Res., 7:145-52 (2001)).

Accumulation of pheophorbide a, a fluorescent ABCG2 substrate (Jonker etal., Proc. Natl. Acad. Sci. USA, 99: 15649-54 (2002) and Robey et al.,Cancer Res., 64: 1242-6 (2004)), formed the basis of the assay forinhibitors of ABCG2 activity (Henrich et al., J. Biomol. Screen, 11:176-83 (2006)). Briefly, NCI-H460/MX20 cells are transferred to blackwall, clear bottom 384-well polylysine-coated assay plates (Corning,Corning, N.Y.) and allowed to attach for several hours. Pheophorbide a(1 μM final concentration) is added immediately followed by compounds orvehicle (DMSO/PBS) control and incubated an additional 18 h. Afterremoval of medium and washing with PBS containing Ca²⁺ and Mg²⁺,fluorescence intensity is read on a Tecan Safire fluorescence platereader in bottom read mode, 395 nm excitation, 670 nm emission. Eachplate has control wells containing 10 μM (final concentration) FTC. Dataare normalized to FTC and reported as % of FTC fluorescence.

FIG. 1 summarizes the activities of the compounds in accordance with anembodiment of the invention in the pheophorbide a accumulation assay.The compounds have activities ranging from 60-105% of the activity ofFTC when evaluated at 10 μM. In this assay, IC₅₀ for FTC was 0.8 μM(Henrich et al., 2006, vide supra). The data also are summarized inTable 1 below.

EXAMPLE 2

This example demonstrates an assay to determine the ability of compoundsto sensitize cancer cells to killing by mitoxantrone in accordance withan embodiment of the invention.

The ability of compounds to sensitize NCI-H460/MX20 cells to killing bymitoxantrone is assessed as described in Henrich et al. (J. Biomol.Screen, 11: 176-83 (2006)). ABCG2 over-expressing cells or parentalcells are treated with mitoxantrone in the presence or absence of 10 μMcompound (or 1 μM FTC) and cell numbers assessed after 2 d by an XTTassay (Scudiero et al., Cancer Res., 48: 4827-33 (1988)). Final DMSOconcentration is 0.2% (v/v).

FIG. 2 illustrates each compound's ability to restore mitoxantronesensitivity to cells overexpressing ABCG2. Unselected NCI-H460 cells aresensitive to killing by mitoxantrone. After 2 days in the presence of 30μM mitoxantrone, cell numbers are 21.6±1.6% (sd) of control (vehicle).None of the compounds tested are significantly cytotoxic againstparental cells (data not shown). Cells selected for ABCG2 overexpression(NCI-H460/MX20) are significantly more resistant to mitoxantrone (see“PBS” column in FIG. 2). After mitoxantrone treatment, NCI-H460/MX20cell number is 56.1±2.1% (sd) of control. In the presence of 1 μM FTC,this number is further reduced to 24.5±1.5% (sd). Similar effects areseen with other tested compounds (at 10 μM). None of the compounds alonecauses significant cell killing in the NCI-H460/MX20 subline (FIG. 2).The data also are summarized in Table 1 below.

EXAMPLE 3

This example demonstrates that compounds in accordance with anembodiment of the invention can inhibit ABCG2-mediated transport usingBODIPY-prazosin as a substrate (Robey et al., Br. J. Cancer, 89: 1971-8(2003)). This example also demonstrates that exemplary compounds inhibitMRP1-mediated calcein efflux (Robey et al., 2003, vide supra and Alvarezet al., Mol. Pharmacol., 54: 802-14 (1998)).

Transfected HEK293 cells expressing ABCG2, Pgp, or MRP1 are trypsinizedand incubated in complete medium (phenol red-free Richter's medium with10% FCS and penicillin/streptomycin) containing 200 nM BODIPY-prazosin,0.5 μg/ml rhodamine 123 or 200 nM calcein AM, respectively, in thepresence or absence of the desired concentration of inhibitor for 30 minat 37° C. The positive controls for inhibition of ABC transporters are10 μM FTC for ABCG2, 3 μg/ml valspodar for Pgp and 25 μM MK-571 forMRP1. Cells are then washed and incubated in substrate-free mediumcontinuing with or without inhibitor for 1 h.

In this assay, compounds of the invention are active in inhibitingBODIPY-prazosin efflux (1.4- to 5.9-fold BODIPY-prazosin accumulation ascompared to 3.5-fold for FTC). Compounds: NSC11668, NSC19139, NSC120688,NSC168201, and NSC375985 are selected for further testing. Column 1 ofFIG. 3 shows the results of a dose-response assay with BODIPY-prazosinwith 0.1, 1, or 10 μM of each of the 5 compounds. The data also aresummarized in Table 1 below.

EXAMPLE 4

This example demonstrates that compounds of the invention are ABCG2inhibitors, rather than substrates, in accordance with an embodiment ofthe invention.

Five compounds are examined for their ability to increase surfacestaining of the 5D3 antibody. Ozvegy-Laczka et al. have previouslydemonstrated that, at high dilution, the 5D3 antibody binds more readilyto ABCG2 when ABCG2-transfected cells are incubated with the antibody inthe presence of an ABCG2 inhibitor (J. Biol. Chem., 280: 4219-27(2005)). This is believed to be due to the fact that, at low antibodyconcentrations, 5D3 has a higher affinity for a certain conformationinduced by inhibitors of ABCG2, allowing study by flow cytometry.

The 5D3 shift assay is performed as described by Ozvegy-Laczka et al.with minor modifications. ABCG2-transfected HEK293 cells are trypsinizedand incubated with 5D3 antibody (1:3500, eBioscience, San Diego, Calif.)for 2 h in the presence or absence of 20 μM of each of the compounds or20 μM FTC as a positive control. Cells are subsequently washed andincubated with APC-labeled goat anti-mouse secondary antibody (1:35) for30 min after which the cells are washed and analyzed. FIG. 3, column 2shows that, at 20 μM, all of the compounds tested increase 5D3 bindingand are comparable to 20 μM FTC shown as a positive control. The changein APC fluorescence is quantified for each sample, and the values aregiven in Table 1.

TABLE 1 Summary of effects of compounds in multiple assays PheophorbideCross- a Flow-ABCG2 reactivity IC₅₀ MX BODIPY- IAAP (flow) Compoundtotal^(a) (μM) sensitization^(b) 5D3^(c) prazosin^(d) binding^(e)Pgp^(f) MRP1^(g) NSC11668 84.1 4.5 21.9 4.0 5.9 24.6 0.73 1.2 NSC1913976.6 2.6 30.0 3.6 3.5 33.5 0.77 1.3 NSC120688 67.2 4.3 20.9 3.7 2.0 19.10.84 0.81 NSC168201 104.5 3.9 21.2 3.7 2.6 34.2 0.64 0.81 NSC375985 63.83.7 27.6 3.2 3.6 48.7 0.84 1.8 FTC 100 0.8 24.5 3.7 3.5 32.7 (control)MK571 3.3 (25 μM) (control) valspodar 14.9 (3 μg/mL) (control) DMSO/PBS0 56.1 1.0 1.0 100 (blank) a % of FTC response ^(b)% NCI-H460/MX20 cellsurvival in the presence of compound and mitoxantrone ^(c)5D3 staining:treated/control ratio at 10 μM compound ^(d)BODIPY-prazosin efflux:treated/control ratio at 10 μM compound ^(e)Blocking of ¹²⁵I-IAAPbinding: % of control binding in the presence of 20 μM compound ^(f)Pgpinhibition, rhodamine efflux: treated/control ratio at 10 μM compound^(g)MRP1 inhibition, calcein efflux: treated/control ratio at 10 μMcompound

EXAMPLE 5

This example demonstrates photoaffinity labeling of ABCG2 with[¹²⁵I]-IAAP. Specifically, the example demonstrates the ability ofcompounds in accordance with an embodiment of the invention to inhibit[¹²⁵I]IAAP incorporation into ABCG2 in membranes isolated fromABCG2-overexpressing MCF-7 FLV1000 cells.

ABCG2 expressed in MCF-7 FLV 1000 cells is photo-labeled with[¹²⁵I]-IAAP as described previously (Shukla et al., Biochemistry, 45:8940-51 (2006)). Briefly, crude membranes (1 mg protein/me of MCF-7FLV1000 cells are incubated with 20 μM of the indicated compound for 10min at room temperature in 50 mM Tris-HCl, pH 7.5. 3-6 nM [¹²⁵I]-IAAP(2200 Ci/mmole) (PerkinElmer Life Sciences, Wellesley, Mass.) is added,and the samples are incubated for an additional 5 min under subduedlight. The samples are then exposed to ultraviolet (UV, 365 nm) lightfor 10 min, and the labeled ABCG2 is immunoprecipitated using BXP-21antibody. The radioactivity incorporated into the ABCG2 band isquantified using the STORM 860 PhosphorImager system (MolecularDynamics, Sunnyvale, Calif.) and ImageQuaNT software (MolecularDynamics).

Since IAAP is a photoaffinity analog of prazosin, it is thought to labelthe drug binding site. All 5 compounds tested (at 20 μM) significantlyreduce [¹²⁵I]-IAAP incorporation into ABCG2 (FIG. 4). It is believedthat the decreased binding is due to competition of the test compoundsfor the drug binding site.

EXAMPLE 6

This Example demonstrates that an ABGC2 inhibitor enhances thechemotherapeutic treatment of a camptothecin derivative SN-38 inaccordance with an embodiment of the invention.

Cytotoxicity assays were performed based on the sulforhodamine B assayreported by Skehan et al (JNCI 1990). Cells were plated at a density of10,000 cells/well in 96-well plates and allowed to attach overnight at37° C. in 5% CO₂. The ABGC2 inhibitor compounds were subsequently addedat various concentrations and plates were allowed to incubate for 96 hat 37° C. in 5% CO₂. Cells were then fixed with 50% trichloroaceticacid, washed and allowed to dry.

Plates were then stained with sulforhodamine B solution (0.4%sulforhodamine B w/v in 1% acetic acid) for 30 minutes and washed 3times in 1% acetic acid solution. Sulforhodamine was then solubilizedwith 10 mM Tris Base and optical densities were ready on a plate readerat an absorbance of 570 nm. Each concentration was tested inquadruplicate and controls were performed in replicates of eight.

The ABCG2 inhibitor compounds were used at 1 μM with varying doses ofSN-38, FTC at 5 μM is the positive control for inhibition. The resultsare shown in FIGS. 5A-5F. The IC50 values decrease when the ABCG2transfected cells are exposed to the ABCG2 inhibitor and treated withSN-38. The foregoing shows that the ABCG2 inhibitor enhances thechemotherapeutic treatment of a cancer treatment agent.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-13. (canceled)
 14. A method of enhancing the chemotherapeutictreatment of tumor cells with a chemotherapeutic agent or a method ofincreasing the bioavailability of an ABCG2 substrate drug in a mammal,which method comprises administering to the mammal an effective amountof the chemotherapeutic agent in conjunction with an effective amount ofa compound to inhibit ABCG2 protein, said compound being selected fromthe group consisting of peliomycin (NSC 76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A.
 15. A methodof reducing resistance of a cancer cell to a chemotherapeutic agent or amethod of inhibiting ABCG2 in a mammal, which method comprisesadministering to the mammal an effective amount of a compound selectedfrom the group consisting of peliomycin (NSC 76455),

and any combination thereof, wherein A of NSC306698 is a mono- ordivalent anion and n=1 or 2 depending on the valency of A, whereuponresistance of the chemotherapeutic agent is reduced in the mammal. 16.The method of claim 14, wherein the chemotherapeutic agent is selectedfrom the group consisting of an antimetabolite, a mitotic inhibitor, analkylating agent, a cytotoxic antibiotic, a topoisomerase inhibitor, atyrosine kinase inhibitor, and any combination thereof.
 17. The methodof claim 16, wherein the chemotherapeutic agent is selected from thegroup consisting of mitoxantrone, topotecan, camptothecin, SN-38,irinotecan, flavopiridol, gefitinib, methotrexate, rhodamine,daunomycin, imatinib, doxorubicin, colchincine, vinblastine, paclitaxel,cisplatin, adriamycin, danofloxacin mesylate, docetaxel, and acombination thereof.
 18. A method of inhibiting MRP1 in a mammalafflicted with cancer, which method comprises administering to themammal an effective amount of a compound selected from the groupconsisting of

and any combination thereof, whereupon MRP1 is inhibited in the mammal.19. The method of claim 14, wherein cancer is treated and the cancer isselected from the group consisting of leukemia, a solid tumor, amelanoma, a non-small cell lung cancer tumor, a colon tumor, a prostatetumor, a brain tumor, a lymphoma, a breast tumor, an ovarian tumor, alung tumor, and a stomach tumor.
 20. The method of claim 14, wherein thecompound is selected from the group consisting of

and any combination thereof.
 21. The method of claim 15, wherein thechemotherapeutic agent is selected from the group consisting of anantimetabolite, a mitotic inhibitor, an alkylating agent, a cytotoxicantibiotic, a topoisomerase inhibitor, a tyrosine kinase inhibitor, andany combination thereof.
 22. The method of claim 21, wherein thechemotherapeutic agent is selected from the group consisting ofmitoxantrone, topotecan, camptothecin, SN-38, irinotecan, flavopiridol,gefitinib, methotrexate, rhodamine, daunomycin, imatinib, doxorubicin,colchincine, vinblastine, paclitaxel, cisplatin, adriamycin,danofloxacin mesylate, docetaxel, and a combination thereof.
 23. Themethod of claim 15, wherein cancer is treated and the cancer is selectedfrom the group consisting of leukemia, a solid tumor, a melanoma, anon-small cell lung cancer tumor, a colon tumor, a prostate tumor, abrain tumor, a lymphoma, a breast tumor, an ovarian tumor, a lung tumor,and a stomach tumor.
 24. The method of claim 16, wherein cancer istreated and the cancer is selected from the group consisting ofleukemia, a solid tumor, a melanoma, a non-small cell lung cancer tumor,a colon tumor, a prostate tumor, a brain tumor, a lymphoma, a breasttumor, an ovarian tumor, a lung tumor, and a stomach tumor.
 25. Themethod of claim 17, wherein cancer is treated and the cancer is selectedfrom the group consisting of leukemia, a solid tumor, a melanoma, anon-small cell lung cancer tumor, a colon tumor, a prostate tumor, abrain tumor, a lymphoma, a breast tumor, an ovarian tumor, a lung tumor,and a stomach tumor.
 26. The method of claim 18, wherein cancer istreated and the cancer is selected from the group consisting ofleukemia, a solid tumor, a melanoma, a non-small cell lung cancer tumor,a colon tumor, a prostate tumor, a brain tumor, a lymphoma, a breasttumor, an ovarian tumor, a lung tumor, and a stomach tumor.
 27. Themethod of claim 15, wherein the compound is selected from the groupconsisting of

and any combination thereof.
 28. The method of claim 16, wherein thecompound is selected from the group consisting of

and any combination thereof.
 29. The method of claim 17, wherein thecompound is selected from the group consisting of

and any combination thereof.